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Plant Protection Department, University of Yu
¨zu
¨ncu
¨Y
ı
l, Van, Turkey
Biological Control of Fusarium Wilt in Tomato Caused by Fusarium oxysporum
f. sp. lycopersici by AMF Glomus intraradices and some Rhizobacteria
A.A. AkkopruAkko
¨pru
¨andand S.S. DemirDemir
Authorsaddress: Faculty of Agriculture, Plant Protection Department, University of Yu
¨zu
¨ncu
¨Yıl, Van, Turkey
(correspondence to S. Demir. E-mail: semrademir@yyu.edu.tr)
Received March 28, 2005; accepted July 7, 2005
Keywords: biological control, fluorescent rhizobacteria, arbuscular mycorrhizal fungi, Fusarium oxysporum f. sp. lycopersici,
tomato
Abstract
In the present study, the effects of the arbuscular my-
corrhizal fungus (AMF) Glomus intraradices Schenck
& Smith and four rhizobacteria (RB; 58/1 and D/2:
Pseudomonas fluorescens biovar II; 17: P. putida; 21:
Enterobacter cloacae), which are the important mem-
bers of the rhizosphere microflora and biological con-
trol agents against plant diseases, were examined in
the pathosystem of Fusarium oxysporum f. sp. lycoper-
sici [(Sacc) Syd. et Hans] (FOL) and tomato with
respect to morphological parameters (fresh and dry
root weight) and phosphorous (P) concentration in the
roots. Treatments with single and dual inoculation
with G. intraradices and RB strains reduced disease
severity by 8.6–58.6%. Individual bacteria inoculations
were more effective than both the single AMF and
dual (G. intraradices + RB) inoculations. In addition,
the RB and G. intraradices enhanced dry root weight
effectively. Significant increases in root weights were
recorded particularly in the triple inoculations com-
pared with single or dual inoculations. Compared with
the non-treated controls all biological control agents
increased P-content of treated roots of plants. Colon-
ization with RB increased especially in triple
(FOL + G. intraradices + RB) inoculations whereas
colonization of G. intraradices was significantly
decreased in treatment of FOL + G. intraradices com-
pared with triple inoculations. The results suggest that
suitable combinations of these biocontrol agents may
ameliorate plant growth and health.
Introduction
Fusarium oxysporum f. sp. lycopersici (FOL) is an soil-
borne pathogen causing wilting occasionally accom-
panied with severe yield loss in tomato. Management
of Fusarium wilt is mainly through chemical soil fumi-
gation and resistant cultivars. The broad-spectrum bio-
cides used to fumigate soil before planting
(particularly methyl bromide) are environmentally
damaging (Blancard, 1993). The most cost-effective
and environmentally safe method of control is the use
of resistant cultivars whenever they are available. On
the contrary, breeding for resistance can be very diffi-
cult when no dominant gene is known. In addition,
new races of pathogens overcoming host resistance can
develop. The difficulty in controlling Fusarium wilt
has stimulated the research in biological control inde-
pendently from the recent concern for environmental
protection (Fravel et al., 2003).
Among the biocontrol agents used against Fusarium
wilt of tomato are arbuscular mycorrhizal fungi (AMF;
Caron et al., 1985a,b; Linderman, 1992; O
¨zgo
¨nen
et al., 1999) and some rhizobacteria (Alabouvette et al.,
1985; Bora et al., 1994; Duijff et al., 1999; Chin-A-
Woeng et al., 2000). Both of the micro-organisms are
important members of the rhizosphere and considered
as effective symbionts by protecting the plants from
root rot pathogens, increasing plant growth and ren-
dering plant tolerance to various stress factors. AMF
may limit fungal root diseases and nematodes by
strengthening morphological traits of plants with
some physiological and microbial modifications in the
mycorrhizosphere and by altering the chemical com-
position of plant tissues (Azco
´n-Aguilar and Barea,
1996; Linderman, 2000; Barea et al., 2002). Rhizobac-
teria may also dominate the pathogens by various
mechanisms such as competition, antibiosis and indu-
ced resistance and then positively affect plant growth
with their promoting (PGPR) properties (Kloepper,
2003).
Much research has been conducted to investigate
and to determine associations and interactions between
AMF and RB, and their effects on plant growth and
defence mechanism. Beside synergistic effects (Meyer
and Linderman, 1986; Paulitz and Linderman, 1989;
Gryndler and Vosa
´tka, 1996; Barea et al., 1998;
Edwards et al., 1998; Ravnskov et al., 1999; Sulochana
et al., 2003) some antagonistic interactions (Waschkies
www.blackwell-synergy.com
J. Phytopathology 153, 544–550 (2005)
2005 Blackwell Verlag, Berlin
et al., 1994; Walley and Germida, 1997; So
¨derberg
et al., 2002) were detected among the symbiotic micro-
organisms. Positive effects increasing both plant
growth and their tolerance to pathogens were deter-
mined in some pathosystems where dual inoculations
of both AMF and RB existed (Budi et al., 1999; Berta
et al., 2003; Hazarika and Phookan, 2003).
First, the present study aims at investigating the
effects of single or dual inoculations of AMF, Glomus
intraradices, and of some Gram-negative RB (Pseudo-
monas fluorescens,P. putida and Enterobacter cloa-
ceae), all considered important members of the
rhizosphere as biocontrol agents, on the disease sever-
ity of FOL in tomato, and secondly to detect the dif-
ference in their colonization rates following all possible
interactions among them.
Materials and Methods
AMF+ and AMF)seedling growth
A mixture of sand, pumice and soil (1 : 1 : 1) was
mixed and sterilized together in an autoclave at 121C
and 1 atm for 1.5 h and was used as a growth med-
ium. The composition of growth medium was as fol-
lows: 0.9% organic matter, 0.023% salt, 10.75% lime,
78.2% sand, 8.28% clay, 13.52% loam, 0.017% nitro-
gen, 2.8035 p.p.m. phosphorous (P), 0.9176 p.p.m.
zinc, 7.7502 p.p.m. manganese, 5.2297 p.p.m. iron,
0.9815 p.p.m. copper and 31.044 p.p.m. potassium, pH
adjusted to 8.1. The growth medium was put into the
plastic trays (33 ·48 ·10) disinfected with 10% of
formaldehyde, and the isolate OM/95 (AMF G. intra-
radices; Demir and Onog
ˇur, 1999) in the mixed inocu-
lum form (at the concentration of 75 spores/10 g of
soil) was added as an approximately 2–3 cm layer to
the growth medium where the tomato seeds were to be
sown. The same amount of sterilized growth medium
was put into the control trays instead of the AMF iso-
late. For the surface disinfection, tomato (cv. Super
Marmende) seeds were soaked into the 2% of NaOCl
solution for 5 min and then washed twice with deion-
ized water before sowing them into the growth med-
ium. All trays were placed in a growth chamber under
standard (appropriate) conditions (14 h light at 25C
with 60% relative humidity and 10 h dark at 15C,
60% relative humidity) for 8 weeks. Plants were
watered twice a week with deionized water and 500 ml
of the nutrient solution [containing 720 mg MgSO
4
Æ
7H
2
O, 12.2 mg KH
2
PO
4
, 295 mg Ca(NO
3
)
2
Æ4H
2
O,
240 mg KNO
3
, 0.75 mg MnCl
2
Æ4H
2
O, 0.75 mg KI,
0.75 mg ZnSO
2
ÆH
2
O, 1.5 mg H
3
BO
3
, 0.001 mg
CuSO
4
Æ5H
2
O, 4.3 mg EDDHAFeNa, 0.00017 mg
NaMoO
4
Æ2H
2
O; modified from Vosa
´tka and Gryndler
(1999)] was applied three times into each tray during
the experiment.
Growth medium for plants and pathogen inoculation
The pathogen isolate no. 83 of FOL was obtained
from the Agricultural Faculty of the University of
Adnan Menderes, grown on potato dextrose agar
(PDA), and then produced on the corn meal and sand
culture (Turhan and Grosman, 1987). Pots,
16 ·18 cm in size, disinfected with 10% of NaOCl
and 4–5 cm of pumice were layered into the bottoms
of the pots before adding 5.5 kg of growth medium. A
10
6
cfu/ml suspension of FOL conidia was then added
to the FOL pots 48 h before seedling transplantation.
Preparation of bacteria suspension and its application to roots
Pseudomonas flourescens biovar II (isolate no. 58/1 and
D/2) and E. cloaceae (isolate no. 21/1K) isolated from
the tomato rhizoplane and P. putida (isolate no. 17)
isolated from tobacco rhizoplane were used as fluores-
cent RB in the present study. Analytical Profile Index
(API) ID 32 GN (Biomeriux, Walpole, USA) diagnos-
tic kits were used in the identification of the bacteria.
Trimmed tomato seedling roots were dipped for
30 min into the 10
9
cfu/ml (OD
600nm
¼0.1) concen-
tration of bacterial suspension prepared from cultures
grown on King’s B (KB) medium.
Eight different treatments, each with three plants in
six replications (total 18 plants for each treatment),
were designed in randomized blocks. All pots were
placed in a growth chamber under standard conditions
(15 h light, 25C, 60% relative humidity and 10 h dark
15C, 60% relative humidity) for 10 weeks. Plants
were watered twice a week with deionized water and
diluted with nutrient solution of Gryndler and Vosa
´tka
(1999) which was applied three times into each pot
(200 ml/pot) during the study.
Determination of disease severity caused by FOL, and root
colonization by rhizobacteria and Glomus intraradices
The wilt development on each tomato plant grown in
the growth chamber for 10 weeks was rated by using
the following scale (Bora et al., 2004): 0, no symptoms;
1, <25% of leaves with symptoms; 2, 26–50% of
leaves with symptoms; 3, 51–75% of leaves with symp-
toms; 4, 76–100% of leaves with symptoms. Their dis-
ease indices were calculated based on the following
formula:
Disease index ¼Pðratingnumber of plants rated)
Total number of plants highest rating100
Tomato roots were dyed in order to determine the
existence of G. intraradices by a modified method of
Phillips and Hayman (1970) and the colonization rates
were determined by the Grid-Line Intersect Method
(Giovanetti and Mosse, 1980). Bacterial densities on
roots were assessed according to Geels and Schippers
(1983).
Determination of the P-contents and fresh and dry matter
weights in tomato roots
At the end of the experiment, tomato plants were har-
vested 10 weeks after seed sowing. Plant roots were
separated, dried (70C, 48 h) and weighed. The vana-
date-molybdate-yellow procedure with spectrophotom-
eter was used for P-analysis in tomato roots (Kacar,
1984).
545Biological Control of Fusarium Wilt
Determination of antagonistic effects
In order to determine the antagonistic effects of RB on
FOL, an agar disc of FOL was placed upside down
into the middle of a 9-cm diameter Petri dish contain-
ing PDA and four sterile filter paper discs were placed
in equal distance to the FOL disc, with 10 ll RB sus-
pensions grown in KB culture poured on each paper
disc. Four Petri dishes of each isolate were prepared
and left to grow for 5 days. Differences in diameters of
RB+ and RB)FOL colonies were measured and the
antagonistic inhibition effect of each RB was calcula-
ted by the following formula (Bora and O
¨zaktan,
1998):
Antagonistic inhibitionð%Þ
¼Diameter of RBðÞFOL Diameter of RB(+) FOL
Diameter of RBðÞFOL 100
Determination of siderophore production
The RB strains antagonistic to FOL were plated at
three different points on 9 cm Petri dishes containing
two different KB media, one deficient in ferric ions
(Fe)) and the other having 80 lmFeCl
3
/l (Fe+).
After 48 h, the dishes were sprayed with conidial sus-
pension of FOL (10
5
CFU/ml) and left to grow for
5 days. Then, the antagonistic siderophore effect was
determined with a scale from 0 to 5 (Geels and
Schippers, 1983): with 0, no inhibition; 1, x£2 mm;
2, 2 mm < x<y; 3, 2 mm < x>y;4,y£2 mm;
5, y¼0, whereby xis the zone where the pathogen
was inhibited, yis the zone where the pathogen devel-
oped. Loss of inhibition zones in the FeCl
3
containing
dishes was the indication of competition for the Fe
3+
ions, i.e. the indication of a siderophore effect (Bora
and O
¨zaktan, 1998).
The data were analysed by anova using sas statisti-
cal program (SAS, 1998). Before the analyses were car-
ried out, percentage data of wilt severity were
transformed using the log-transformations. Differences
between treatments were determined by Duncan’s mul-
tiple range test at 5% significant level.
Results
Disease severity
Percentage efficacy of the AMF G. intraradices and the
various RB strains combinations with both single and
dual inoculations against FOL are seen in Table 1.
Both members of the rhizosphere were effective in the
inhibition of FOL and their effects in single and dual
inoculations ranged between 8.6% and 58.6%. While
the single application of G. intraradices inhibited FOL
at the rate of 17.3%, its dual applications, except for
D/2, were more effective than its single applications
(Table 1). On the contrary, single applications of RB
isolates were more effective in the inhibition of FOL
than their dual applications.
P-contents and fresh and dry matter weights in tomato roots
Impacts of the biocontrol agents on the P-content,
and fresh and dry matter weights of tomato roots
were also determined besides their effects on FOL
(Table 2). Increases in these mentioned traits were
detected in all treatments including both biocontrol
agents. There were significant differences among the
treatments. The P-content in the single application
of FOL (1384.1 p.p.m.) was lower than those in
single or dual applications of the biocontrol agents
whose P concentrations ranged from 1546.4 to
2537.9 p.p.m. (Table 2). The most remarkable
result was obtained from the application of FOL
+G. intraradices + D/2 (P. putida) whose dry root
matter content was 107% higher than the control
(Table 2).
Table 1
Percentage efficacy of the Glomus intraradices and of various RB
strains with single and dual inoculations for control Fusarium oxy-
sporum f. sp. lycopersici in tomato
Treatments
Disease
severity (%) Efficacy (%)
b
Control (+)
a
46 a
c
–
G. intraradices 38 b 17.3
58/1 (Pseudomonas fluorescens) 29 c 36.9
21/1K (Enterobacter cloaceae) 32 bc 30.4
17 (P. putida) 19 d 58.6
D/2 (P. fluorescens) 42 a 8.6
G. intraradices + 58/1 (P. fluorescens) 36 b 21.7
G. intraradices + 21/1K (E. cloaceae) 32 bc 30.4
G. intraradices +17(P. putida) 32 bc 30.4
G. intraradices + D/2 (P. fluorescens) 39 b 15.2
a
Control (+), pathogen alone.
b
Percentage of reduction in wilt severity compared with the pathogen
alone.
c
Mean values followed by the same letter are not significantly differ-
ent according to DMRT at 5% significance level.
RB, rhizobacteria; DMRT, Duncan’s multiple range test.
Table 2
Dry and fresh weights and contents of phosphorous (P) content of
tomato plant roots affected by FOL, Glomus intraradices and rhizo-
bacteria treatments
Treatments
Root
Fresh
weight (g)
Dry
weight (g) P (p.p.m.)
Control ()) 7.3 f 0.53 g 941.3 g
FOL 11.3 d 0.77 d 1384.1 f
FOL + G. intraradices 9.8 e 0.65 f 1977.9 b
FOL + 58/1 (P. fluorescens) 10.8 d 0.67 e 1668 d
FOL + 21/1K (E. cloaceae) 13.9 cd 0.72 de 1546.4 e
FOL + 17 (P. putida) 14.4 c 0.78 d 1715.8 c
FOL + D/2 (P. fluorescens) 14.7 c 0.67 e 1811.1 bc
FOL + G. intraradices +
58/1 (P. fluorescens)
17.3 b 0.85 c 1751.5 c
FOL + G. intraradices +
21/1K (E. cloaceae)
13.3 cd 0.72 de 2418.7 a
FOL + G. intraradices +
17 (P. putida)
19.2 a 0.9 b 2537.9 a
FOL + G. intraradices +
D/2 (P. fluorescens)
20.1 a 1.1 a 1727.7 c
Mean values followed by the same letter are not significantly differ-
ent according to DMRT at 5% significance level.
FOL, Fusarium oxysporum f. sp. lycopersici; DMRT, Duncan’s mul-
tiple range test.
546 Akko
¨pru
¨and Demir
Antagonistic and siderophore effects of RB strains in in vitro
The in vitro efficacy of RB strains against FOL was
determined by antibiosis and siderophore tests. At the
end of the incubation period, it was observed that the
FOL growth at the centre of each Petri dish was inhibi-
ted in different levels by the RB strains planted in equal
distances (Table 3). The RB strains inhibited the patho-
gen in vitro at the range from 24% to 32%. While the
highest inhibition was achieved from the strain D/2,
the lowest one was obtained from the strain 21/1K. In
the in vitro study aiming whether this antagonistic
effect was caused by the siderophore mechanism or
not, the RB strains grown in iron-deficient medium
(Fe)) showed the inhibition zones at various levels and
these zones were not observed in iron added medium
(Fe+) except for the isolate D/2 which formed a thin
inhibition region in iron added medium (Table 3).
Colonization rates of G. intraradices and RB strains
Colonization rates of the biocontrol agents, G. intra-
radices and the RB strains are presented in Tables 4
and 5. As seen in Table 4, it was determined that FOL
alone significantly (52.3%) reduced the colonization
level of G. intraradices compared with the non-treated
control, and the FOL treatments accompanied with
the other RB strains reduced the colonization level of
G. intraradices between 17% and 50%. The RB strains
alone had either positive (the strains 21/1K and D/2)
or negative (58/1 and 17) impacts on the colonization
level of G. intraradices (Table 4). The population den-
sities of the RB strains in the tomato roots varied
based on the treatments (Table 5). The colonies were
counted at the 10
4
,10
5
and 10
6
dilution levels of bac-
teria stock suspensions except for the D/2 isolate.
There was an insignificant increase in the colonization
rates of the RB strains coupled with G. intraradices
while there was a decrease in their colonization levels
in case of FOL presence except for isolate 17
(Table 5).
Discussion
In the present study, AMF, G. intraradices, and some
Gram-negative and fluorescent RB, P. fluorescens,
P. putida and E. cloaceae, isolated from the rhizoplane
of solanaceous plants were employed against the
important soil-borne pathogen of tomato, FOL. Both
biocontrol agents (either AMF or RB), which are
important members of the rhizosphere population, are
very efficient and successful in the inhibition of especi-
ally root rot diseases (Kloepper et al., 1993; Azco
´n-
Aguilar and Barea, 1996; Chin-A-Woeng et al., 2000;
Berta et al., 2003). For this purpose, both single and
dual applications of these biocontrol agents were
employed against FOL in the present study and were
found to be effective in the inhibition of FOL with up
to 58% although the success rate varied among the
different treatments (Table 1). The results obtained in
the present study are in agreement with the results
of other researchers (Vandenbergh et al., 1983;
Caron et al., 1985a,b, 1986; O
¨zgo
¨nen et al., 1999;
Ramamoorthy et al., 2002).
Table 3
Siderophore effect and antagonistic inhibition values (%) of rhizo-
bacteria (RB) strains against Fusarium oxysporum f. sp. lycopersici in
vitro
RB isolates
Siderophore effect
Antagonistic
inhibition (%)
Scale values
a
Medium with
no iron (Fe))
b
Medium with
iron (Fe+)
b
D/2 (P. fluorescens)2 0 24
21/1K (E. cloaceae)1 0 32
17 (P. putida)1029
58/1 (P. fluorescens)1 0 25
a
0–5 scale: 0, no inhibition; 1, x£2 mm; 2, 2 mm < x<y;3,
2mm<x>y;4,y£2 mm; 5, y¼0, whereby xis the zone
where the pathogen was inhibited, yis the zone where the pathogen
developed.
b
Scale was determined according to the colony development meas-
ured in 9-cm Petri containing King’s B medium.
Table 4
Colonization rates of Glomus intraradices on tomato roots inoculated
with FOL and various rhizobacteria
Treatments
AMF root
colonization (%)
Difference
(%)
b
Control (G. intraradices)
a
84 b
c
0.00
G. intraradices + FOL 40 e ()) 52.3
G. intraradices + 58/1 (P. fluorescens)82b()) 2.3
G. intraradices + 21/1K (E. cloaceae) 92 a (+) 9.5
G. intraradices +17(P. putida)74c()) 11.9
G. intraradices + D/2 (P. fluorescens) 85 b (+) 1.1
G. intraradices + 58/1
(P. fluorescens) + FOL
69 cd ()) 17.8
G. intraradices + 21/1K
(E. cloaceae) + FOL
42 de ())50
G. intraradices +17
(P. putida) + FOL
54 d ()) 35.7
G. intraradices + D/2
(P. fluorescens) + FOL
54 d ()) 35.7
a
Control (G. intraradices), G. intraradices alone.
b
Percentage of increase (+) and decrease ()) rate in root coloniza-
tion compared with the G. intraradices alone.
c
Mean values followed by the same letter are not significantly differ-
ent according to DMRT at 5% significant level.
FOL, Fusarium oxysporum f. sp. lycopersici; AMF, arbuscular
mycorrhizal fungus; DMRT, Duncan’s multiple range test.
Table 5
Colonization rates of RBs on tomato roots inoculated with FOL
and Glomus intraradices (CFU/plant)
RB
strains
Treatments
Control
(RB) FOL + RB
G. intraradices
+RB
G. intraradices
+ RB + FOL
58/1 1 ·10
6
2.9 ·10
5
1·10
6
6.8 ·10
6
21/1K 1.3 ·10
6
1·10
6
2.1 ·10
6
1.6 ·10
6
17 1 ·10
5
3.2 ·10
6
1.3 ·10
5
1·10
6
D/2 8.9 ·10
5
–– –
FOL, Fusarium oxysporum f. sp. lycopersici; RB, rhizobacteria.
547Biological Control of Fusarium Wilt
In some of the studies the disease inhibition by
AMF was linked to their ameliorative effects for plant
nutrients especially for P-content (Caron et al., 1986;
O
¨zgo
¨nen et al., 1999). However, Bo
¨dker et al. (1998)
reported that P-content alone was not sufficient for
restricting the disease development. Hassan Dar et al.
(1997) determined that the disease severity diminished
in parallel to the reduction in the numbers of F. solani
propagules that are around the root rhizosphere colon-
ized with AMF. Therefore, in the present study we
have presumed that the disease inhibition of AMF
might not be completely related to the increase in
P-content although there is a significant increase in
P-contents and dry weights of roots (Table 2). It has
been thought that beside the plant nutrient uptake the
competition for space and nutrients, changes in root
system, mycorrhizosphere effect and the activation of
plant defence mechanisms are responsible for disease
inhibition by AMF (Linderman, 1994; Azco
´n-Aguilar
and Barea, 1996; Demir and Akko
¨pru
¨, 2005). The RB
strains alone were also very effective in the inhibition
of FOL (Table 2). Vandenbergh et al. (1983) and
Ramamoorthy et al. (2002) stated that P. fluorescens
stimulated the defence mechanisms in plants and made
the plants more resistant to FOL. The same research-
ers acknowledged that the competition for iron
(siderophore production) was used for the inhibition
of FOL. In the present study, the strain D/2 (P. fluo-
rescens) had a significant siderophore effect on FOL,
while the other strains (17: P. putida, 58/1: P. fluores-
cens and 21/1K: E. cloaceae) had only small inhibition
zones against the pathogen on the iron-deficient med-
ium (Table 3). Besides having poor siderophore effects,
these isolates might also have been inhibited by the
pathogen due to space competition (Chin-A-Woeng
et al., 2000; Lagopodi et al., 2002). However, the inter-
esting result was that strain D/2 having the highest
inhibition zone in vitro became the weakest isolate
in vivo for the inhibition of FOL (Table 3). Discrep-
ancy between the in vivo and in vitro results might be
associated with the various interactions caused by dif-
ferent environments and with differentiations in the
traits of micro-organisms affected by the environment
(Bora and O
¨zaktan, 1998). Dual applications of G. in-
traradices and the RB isolates were also effective in
inhibiting FOL in the range of 15.2–30.4% (Table 1).
The dual applications of G. intraradices with the RB
isolates, except for D/2, were especially more effective
than its single application (Table 1). It has been repor-
ted that dual application of AMF and bacterium
inhibited the pathogen more efficiently (Budi et al.,
1999; Sulochana et al., 2003) and caused fewer plant
deaths (Hazarika and Phookan, 2003) than single
applications. Moreover, it has been stated that dual
applications of both AMF and rhizobacteria (PGPR)
are becoming efficient by inhibiting parasitic growth of
any pathogen on the plant root (Berta et al., 2003).
Their mutual establishment also improves plant root-
ing and enhances plant growth and nutrition (Azco
´n-
Aguilar and Barea, 1996).
AMF and RB, as the most important symbionts
of rhizosphere, have shown stimulating (Meyer and
Linderman, 1986; Andrade et al., 1997; Barea et al.,
1998; Edwards et al., 1998) or inhibiting (Walley and
Germida, 1997; So
¨derberg et al., 2002) effects on each
other or on the growth of plants and pathogens. This
was also confirmed in our study. The RB strains either
increased or reduced the colonization of G. intraradices
in dual applications of both symbionts, but no clear
inhibition was observed (Table 4). The RB strains
21/1K and D/2 were found to be more stimulating for
the AMF colonization. Especially 21/1K increased the
AMF colonization at the rate of 9.5% (Table 4). It
has been reported that the species having stimulating
effects on AMF colonization are called mycorrhiza
helper bacteria(Barea et al., 1998) and these promote
root colonization by stimulating the germination of
AMF spores and mycelial development (Meyer and
Linderman, 1986; Edwards et al., 1998; Sood, 2003).
The inhibiting effects of RB strains on AMF coloniza-
tion are thought to be related to the secretion of anti-
microbial substances (Walley and Germida, 1997; Mar
Va
´zquez et al., 2000). Dual applications of AMF and
RB had also positive effects on the population densi-
ties of the RB isolates; the bacterial colonization
increased compared with single applications (Table 5).
AMF influence the microbial populations in the rhizos-
phere of plants, and may especially have selective
effects on the bacteria residing in the hyphosphere
(microenvironment surrounding the mycorrhizal
mycelium itself) and increase their populations
(Andrade et al., 1997; Mansfeld-Giese et al., 2002).
The change in the densities of bacteria populations
found in the roots colonized by AMF is linked to the
changes in the qualities and quantities of the root
exudates induced by AMF colonization and some
subsequent differentiations in the root membrane
permeability (Waschkies et al., 1994; Sood, 2003).
Linderman (2000) reported that some antagonistic
interactions might occur among the micro-organisms
inhibiting the same pathogen. It is not clear how AMF
root colonization may be affected by soil-borne patho-
gens. Some reports stated that AMF root colonization
was not affected by pathogens such as Fusarium spp.
(Caron et al., 1986; O
¨zgo
¨nen et al., 1999) and some
confirmed that AMF was negatively affected by the
pathogens in different pathosystems and the root col-
onization was reduced (Zambolim and Schenck, 1983;
Hassan Dar et al., 1997). In the present study, it was
observed that FOL negatively affected the AMF coloni-
zation and reduced it by 52.3% (Table 4). In contrast,
FOL negatively affected two of the RB isolates (58/1
and 21/1K), whereas the isolate 17 (P. putida) was
significantly stimulated by FOL (Table 5). Recent
studies have demonstrated that underlying mechanisms
of these differential interactions are still unclear
(Johansson et al., 2004). However, it has been hypo-
thesized that these effects may be related to the
species and varieties of bacteria and the conditions in
548 Akko
¨pru
¨and Demir
the rhizosphere (Siddiqui and Shaukat, 2002; Anjair
et al., 2003).
The present study demonstrates that the two groups
of rhizosphere organisms, AMF and RB, can coexist
without exhibiting adverse effects on each other. More-
over, it is concluded that suitable combinations of
these biocontrol agents may increase the plant growth
and resistance to pathogens. In future studies, there-
fore more detailed investigations of the relationships in
various pathosystems and of the interactions between
the micro-organisms and the host plant are needed for
further developing the biocontrol of the related
diseases.
Acknowledgements
This work is part of a MSc thesis by Ahmet Akko
¨pru
¨under the
supervision of Semra Demir. It was supported by Scientific Research
Foundation of Yu
¨zu
¨ncu
¨Yıl University (2002-ZF-047). Authors also
thank Dr Suat S¸ ensoy for his presubmission review of the manu-
script.
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